Recombinant factor x with no glycosylation and method for preparing the same

ABSTRACT

A Factor X (hereinafter referred to as “FX”) with a high activity is provided. The present invention relates to a method for efficiently preparing a recombinant, two-chain FX which comprises intervening glycosylation at such an amino acid sequence that is essential for glycosylation in FX to thereby allow for expression of a recombinant FX with no glycosylation, and the recombinant FX with no glycosylation obtained by said method.

TECHNICAL FIELD

The present invention relates to a recombinant Factor X (hereinafteralso referred to as “FX”) that may be expressed as a two-chain proteinwhere a high enzymatic activity and an efficient recombinant expressionare possible. Specifically, the present invention relates to a methodfor efficiently preparing a two-chain, recombinant FX by interveningglycosylation at such an amino acid sequence that is essential forglycosylation, said amino acid sequence being present within anactivation peptide domain of FX, to allow for expression of arecombinant FX with no glycosylation for improving expressionefficiency.

BACKGROUND ART

It is widely known that FX is a vitamin K dependent blood coagulationfactor. Like the other vitamin K dependent factors, FX possesses a Gladomain consisting of 11 γ-carboxyglutamic acids (hereinafter alsoreferred to as “Gla”) in the amino acid sequence beginning from theN-terminal to the 39th residue (Non-patent reference 1). In vitro, FX isconverted into activated Factor X (hereinafter also referred to as“FXa”) by an activated Factor VII (hereinafter also referred to as“FVIIa”) or an activated Factor IX (hereinafter also referred to as“FIXa”). FX is used for the treatment of hemophilia patients withinhibitor where an inhibitor to FVIII or FIX is produced as aconsequence of substitution therapy with said FVIII or FIX.

Human FX, in the course of its biosynthesis, is subject toposttranslational modification such as generation of Gla, cleavage of aprepro sequence (the sequence of FX after this cleavage is shown in SEQID NO: 1), β-hydroxylation of aspartic acid at position 63 in SEQ ID NO:1, asparagine-type glycosylation at positions 181 and 191,serine/threonine-type glycosylation at positions 159, 171 and 443, andthe like. It is thought that FX, after being synthesized as asingle-chain protein, is subject to limited degradation with furin, asignal peptidase, at the cleavage motif Arg-Arg-Lys-Arg at positions 139to 142 in SEQ ID NO: 1 to thereby secrete a two-chain protein.

For expression of a recombinant FX, the expression as a two-chainprotein is the most important. It is known that a recombinant expressionfrom an expression vector to which cDNA (SEQ ID NO: 3) encoding theamino acid sequence of FX (the amino acid sequence of FX including theprepro sequence is shown in SEQ ID NO: 2) is simply ligated results inexpressed products, most of which are secreted into culture supernatantas a single-chain protein and which have a low specific activity.

In general, in recombinant factors, their expression level is often thematter. For genetic recombination of Factor X in the present invention,in addition to its expression level, the process for generating atwo-chain protein was thought to be a rate-determining (Non-patentreference 2). In Non-patent reference 2, Himmelspach et al. co-expressedFX with furin so as to promote generation of a two-chain protein with ashigh an expression level of FX as 120 μg/ml or more but with a lowactivity of 25%.

-   Non-patent reference 1: Journal of Thrombosis and Haemostasis, 3:    2633-2648 (2005)-   Non-patent reference 2: Thrombosis Research 97: 51-67 (2000)

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by theInvention

A problem to be solved by the present invention is to prepare andprovide a recombinant, two-chain FX with a high activity.

Means for Solving the Problems

Under the circumstances, the present inventors have assiduouslyinvestigated so as to prepare a recombinant, two-chain FX with a highactivity, and as a result, having regard to a sugar chain of FX, havesucceeded in preparing a secreted, two-chain FX by interveningglycosylation, to thereby complete the present invention.

Thus, the present invention includes the following (1) to (13):

(1) A method for efficiently preparing a recombinant, two-chain Factor X(hereinafter also referred to as “FX”) which comprises interveningglycosylation at such an amino acid sequence that is essential forglycosylation in FX to thereby allow for expression of a recombinant FXwith no glycosylation.(2) The method of (1) above wherein the recombinant FX with noglycosylation is a recombinant FX with no glycosylation at asparagine atposition 181 (Asn181) and/or asparagine at position 191 (Asn191) in SEQID NO: 1.(3) The method of (2) above wherein the recombinant FX with noglycosylation at Asn181 and/or Asn191 is obtained by substituting Asn181and/or Asn191 with a protein-constituting amino acid other than Asn.(4) The method of (2) above wherein the recombinant FX with noglycosylation at Asn181 and/or Asn191 is obtained by substitutingthreonine at position 183 (Thr183) and/or threonine at position 193(Thr193) with a protein-constituting amino acid other than threonine(Thr) or serine (Ser).(5) The method of (1) above wherein intervening glycosylation at such anamino acid sequence that is essential for glycosylation in FX is carriedout by adding an inhibitor to glycosyltransferase during cell culture.(6) The method of (5) above wherein the inhibitor to glycosyltransferaseis tunicamycin, RNAi, or an antisense DNA.(7) The method of (1) above wherein intervening glycosylation at such anamino acid sequence that is essential for glycosylation in FX is carriedout by using a glycosyltransferase-deficient cell strain as a host cell.(8) A recombinant FX with no glycosylation obtained by the method of anyof (1) to (7) above.(9) A gene fragment comprising a nucleotide sequence encoding therecombinant FX with no glycosylation of (8) above.(10) An expression vector comprising the gene fragment of (9) above.(11) A transformed cell in which the expression vector of (10) above isintroduced.(12) A pharmaceutical composition comprising the recombinant FX with noglycosylation of (8) above as an active ingredient.(13) A therapeutic agent effective for the treatment of a hemophiliapatient comprising the pharmaceutical composition of (12) above.

MORE EFFICACIOUS EFFECTS THAN PRIOR ART

The recombinant FX with no glycosylation obtained in accordance with thepresent invention may efficiently be expressed as a two-chain protein.Accordingly, the recombinant FX of the present invention may be used asa medicament quite useful for substitution therapy to hemophiliapatients, in particular, those patients possessing an inhibitor.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of Western blot with antibodies to variousrecombinant FXs obtained by the present invention, showing expressionpatterns of the recombinant FXs in culture supernatant with BHK cells asa host. Lane 1: Variant supernatant (D63A) where aspartic acid atposition 63 is substituted with alanine; Lane 2: Variant supernatant(T159A) where threonine at position 159 is substituted with alanine;Lane 3: Variant supernatant (T171A) where threonine at position 171 issubstituted with alanine; Lane 4: Variant supernatant (N181A) whereasparagine at position 181 is substituted with alanine; Lane 5: Variantsupernatant (N191A) where asparagine at position 191 is substituted withalanine; Lane 6: Variant supernatant (T443A) where threonine at position443 is substituted with alanine; Lane 7: Wild-type recombinant FXsupernatant; and Lane 8: Negative control supernatant.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have focused on sites of posttranslationalmodification such as glycosylation and carried out amino acidsubstitution in FX to successfully prepare a recombinant FX having ahigh enzymatic activity. The recombinant FX according to the presentinvention is explained in detail hereinbelow.

In general, asparagine-type glycosylation is initiated at the site:Asn-X-Thr/Ser where X is any amino acid other than Pro by variousglycosyltransferases in the endoplasmic reticulum followed by furthermodification in the Golgi body (Molecular Biology of The Cell 2ndedition, Chapter 8, Bruce Alberts et al. Garland Publishing, Inc.).Accordingly, the Asn or Thr/Ser may be substituted with other aminoacids for intervening glycosylation to allow for efficient generation ofa two-chain, recombinant FX and expression of the recombinant FX havinga high activity.

For an amino acid to be used the purpose of amino acid substitution,alanine (Ala) is selected herein by way of example but any amino acidmay be used insofar as it does not cause any significant disturbancesuch as loss of an enzymatic activity.

The variant with glycosylation being intervened may be obtained by usinggenetic recombination technique. A host cell is preferably a eukaryotesuch as animal cells. The variant of the present invention may beobtained by incorporating cDNA encoding an amino acid sequence of thevariants into a suitable expression vector, transfecting a host cellwith the vector, cloning cells that express the desired gene, culturingthe obtained stable culture cells, followed by purification.

In addition to amino acid substitution, asparagine-type glycosylationmay also be intervened by adding an inhibitor to glycosyltransferasesuch as tunicamycin to a culture medium of cells (Current Protocols inProtein Science VOL. 2 Chapter 12, John E. Coligan et al. John Wiley &Sons, Inc.).

Furthermore, intervention of asparagine-type glycosylation may also bepossible by intervening expression of glycosyltransferase by the use ofRNAi, an antisense DNA, and the like.

Besides, the glycosylation may also be intervened by using aglycosyltransferase-deficient cell strain as a host cell.

The FX variant of the present invention may be formulated into apharmaceutical formulation for use in therapy, diagnosis, and the like.For preparing a formulation for intravenous administration, thecomposition may usually be dissolved in an aqueous solution containing aphysiologically acceptable substance, e.g. sodium chloride, glycine,etc. and having a buffered pH acceptable to physiological conditions. Toensure long-term stability, a lyophilized form of the formulation mayalso be considered as a final dosage form. Guidelines for a compositionfor intravenous administration are established by government regulationssuch as “Minimum Requirements for Biological Products”.

Specific use of a pharmaceutical composition comprising the FX variantof the present invention may include the use for the treatment ofhemophilia patients with inhibitor where an inhibitor to FVIII or FIX isproduced as a consequence of substitution therapy with said FVIII orFIX.

EXAMPLE

The present invention is explained by means of the following Examplesbut should not be construed to be limited thereto. In Examples, thevariants were those expressed in culture supernatant of animal cells(BHK). Reagents for genetic recombination were purchased from TAKARASHUZO CO., LTD., TOYOBO, Perkin Elmer Applied, and New England Biolabsunless otherwise instructed.

Example 1 Cloning of FX cDNA

A human liver cDNA library (OriGene Technologies) was purchased. Basedon a cDNA sequence (shown in SEQ ID NO: 3) encoding an amino acidsequence of FX comprising a prepro sequence as known in literatures(Molecular Basis of Thrombosia and Hemostasis edited by K. A. High andH. R. Roberts, Marcel Dekker, Inc. 1995), PCR was performed using asense primer for FX synthesis with addition of SalI site (FX-S);GGCGTCGACCCACCATGGATGGGGCGCCCACTGCACCTC (SEQ ID NO: 10) and an antisenseprimer with addition of XhoI site (FX-AS): CTCGAGTTATCACTTTAATGGAGAGGA(SEQ ID NO: 11) and the PCR products were cloned into a commerciallyavailable cloning vector pCRII (Invitrogen). DNA sequencing wasconducted as ordinary to confirm the presence of the sequence known inthe literatures.

Example 2 Preparation of FX Expression Vector

The expression vector pCAGG (Japanese Patent No. 2824434) was digestedwith SalI and was ligated thereto the DNA fragment prepared in Example 1which comprises the sequence encoding FX and has been cleaved withSalI/XhoI. E. coli JM109 cells were transformed with the resultingvector and cultured on LB agar medium supplemented with ampicillin toselect transformed E. coli cells. Colonies as observed were culturedovernight on a commercially available medium and the expression plasmidof interest was extracted and purified to prepare “pCAGFX”. DNAsequencing was conducted for the expression vector to confirm thepresence of the gene sequence of interest.

Example 3 Introduction of Mutation

The FX cDNA as described in Example 1 was digested with restrictionenzymes SalI/XhoI and the fragments were extracted and cloned into pKFvector contained in Site-Directed Mutagenesis kit Mutan-Super Express Kmmanufactured by TaKaRa. 5′-Phospohrilated synthetic DNA primers(Table 1) were prepared in accordance with the annex of the kit and wereused to produce six variants in total with alanine substitution at thecharged amino acid of interest. For all the variants, the sequence wasconfirmed with an automatic DNA sequencer (Beckman Coulter K. K.).

TABLE 1  Primer Sequence of primer SEQ ID NO: D63A-SAAATGTAAAGCCGGCCTCGGG 12 T159A-S GACAGCATCGCATGGAAGCCA 13 T171A-SCTGGACCCCGCCGAGAACCCC 14 N181A-S CTTGACTTCGCCCAGACGCAG 15 N191A-SGGCGACAACGCCCTCACCAGG 16 T443A-S GAGGTCATAGCGTCCTCTCCA 17

Example 4 Preparation of Variant Expression Vector

The expression vector pCAGG (Japanese Patent No. 2824434) was digestedwith SalI and was ligated thereto the fragment prepared in Example 3which comprises the point mutation in the sequence encoding FX and hasbeen cleaved with SalI/XhoI. E. coli JM109 cells were transformed withthe resulting vector and cultured on LB agar medium supplemented withampicillin to select transformed E, coli cells. Colonies as observedwere cultured overnight on a commercially available medium and theexpression plasmids of interest were extracted and purified.

Example 5 Expression of Variants in Culture Supernatant

With the variant FX expression vectors obtained in Example 4, genetransfection was performed to BHK cells using a commercially availablelipofectin reagent (TransIT; TaKaRa) and transient expression culturesupernatant was collected on Day 3 after the transfection. Thesupernatant was concentrated 10-fold with Centricon YM-10 (Millipore)and the expression level was quantified with a commercially availableELISA kit (Funakoshi Co., Ltd.) for FX quantification (Table 2).

Example 6 Measurement of Coagulation Activity of Variants

A coagulation activity of the variants was measured as ordinary by acoagulation approach using FX deficient plasma. Each of the purifiedvariants were diluted to 10 ng/ml to 10 μg/ml with a Veronal buffer(28.5 mM sodium barbital, 125.6 mM NaCl, pH 7.35), mixed with FXdeficient plasma, and after incubation at 37° C., added with an APTTreagent and then with 0.025 M calcium chloride solution to initiate acoagulation reaction. A coagulation time was measured and a coagulationactivity was calculated from a standard curve and a dilution rate (Table2). In addition, the coagulation activity was converted into theactivity per protein level (Example 5; measured by ELISA) to give aspecific activity (Table 2). As a result, among the FX variants of thepresent invention were those variants (N181A, N191A) that showed ahigher expression level and a higher coagulation activity than those ofFX from plasma or a wild-type recombinant FX (Table 2).

TABLE 2 Coagulation Expression activity Specific Ratio to FX Variantlevel*² (ng/mL) activity*³ from plasma*⁴ A*¹ — — 1   1 (3.0) B*¹ 568 1890.33 0.33 (1) C*¹ 361 49 0.14 0.14 (0.42) D*¹ 504 302 0.60 0.60 (1.82)E*¹ 568 29 0.05 0.05 (0.15) F*¹ 959 517 0.54 0.54 (1.64) G*¹ 1036 25542.47 2.47 (7.48) H*¹ 547 358 0.65 0.65 (1.97) *¹A: Standard FX fromplasma; B: Wild-type recombinant FX; C: D63A variant (SEQ ID NO: 4); D:T159A variant (SEQ ID NO: 5); E: T171A variant (SEQ ID NO: 6); F: N181Avariant (SEQ ID NO: 7); G: N191A variant (SEQ ID NO: 8); H: T443Avariant (SEQ ID NO: 9) *²ELISA (ng/mL) *³Coagulation activity/ELISA*⁴(Ratio to wild-type recombinant)

Example 7 Western Blot of Recombinant FXs

The enzymes of the present invention were detected by Western blot usingordinary procedures (Current Protocols in Molecular Biology: Chapter 10analysis of proteins, Chapter 11 immunology, and the like).Specifically, the expression of the recombinant FXs was confirmed bySDS-PAGE under reduced conditions of culture supernatant of BHK cellsexpressing the variant obtained in Example 5, and after transfer to PVDFmembrane, reaction with an anti-human FX monoclonal antibody (FIG. 1).As a result, it was apparent that the variants with mutations at theasparagine-type glycosylation site, Asn181 and Asn191, werepredominantly expressed as a two-chain protein.

1-13. (canceled)
 14. A process for preparing a recombinant Factor X withinhibited glycosylation, wherein the process comprises inhibitingglycosylation of asparagine at position 181 (N181) of SEQ ID NO: 1,inhibiting glycosylation of asparagine at position 191 (N191) of SEQ IDNO: 1, or inhibiting glycosylation of asparagine at position 181 (N181)and position 191 (N191) of SEQ ID NO:
 1. 15. The process according toclaim 14, wherein the process comprises inhibiting glycosylation ofasparagine at position 181 (N181) of SEQ ID NO:
 1. 16. The processaccording to claim 15, wherein the process comprises substituting aprotein comprising an amino acid other than asparagine for asparagine atposition 181 (N181) of SEQ ID NO:
 1. 17. The process according to claim15, wherein the process comprises substituting a protein comprising anamino acid other than threonine or serine for threonine at position 183(T183) of SEQ ID NO:
 1. 18. The process according to claim 14, whereinthe process comprises inhibiting glycosylation of asparagine at position191 (N191) of SEQ ID NO:
 1. 19. The process according to claim 18,wherein the process comprises substituting a protein comprising an aminoacid other than asparagine for asparagine at position 191 (N191) of SEQID NO:
 1. 20. The process according to claim 18, wherein the processcomprises substituting a protein comprising an amino acid other thanthreonine or serine for threonine at position 193 (T193) of SEQ IDNO:
 1. 21. The process according to claim 14, wherein the processcomprises inhibiting glycosylation of asparagine at position 181 (N181)and position 191 (N191) of SEQ ID NO:
 1. 22. The process according toclaim 21, wherein the process comprises substituting a proteincomprising an amino acid other than asparagine for asparagine atposition 181 (N181) and position 191 (N191) of SEQ ID NO:
 1. 23. Theprocess according to claim 21, wherein the process comprisessubstituting a protein comprising an amino acid other than threonine orserine for threonine at position 183 (T183) and position 193 (T193) ofSEQ ID NO:
 1. 24. A recombinant Factor X with inhibited glycosylation,wherein the recombinant Factor X is obtained by a process comprisinginhibiting glycosylation of asparagine at position 181 (N181) of SEQ IDNO: 1, inhibiting glycosylation of asparagine at position 191 (N191) ofSEQ ID NO: 1, or inhibiting glycosylation of asparagine at position 181(N181) and position 191 (N191) of SEQ ID NO:
 1. 25. The recombinantFactor X with inhibited glycosylation according to claim 24, wherein therecombinant Factor X is obtained by a process comprising inhibitingglycosylation of asparagine at position 181 (N181) of SEQ ID NO: 1 bysubstituting a protein comprising an amino acid other than asparaginefor asparagine at position 181 (N181) of SEQ ID NO: 1, substituting aprotein comprising an amino acid other than threonine or serine forthreonine at position 183 (T183) of SEQ ID NO: 1, or both.
 26. Acomposition comprising: the recombinant Factor X according to claim 25;and a physiologically acceptable excipient.
 27. A method of treatinghemophilia, wherein the method comprises administering to a subject inneed thereof an effective amount of the composition according to claim26.
 28. The recombinant Factor X with inhibited glycosylation accordingto claim 24, wherein the recombinant Factor X is obtained by a processcomprising inhibiting glycosylation of asparagine at position 191 (N191)of SEQ ID NO: 1 by substituting a protein comprising an amino acid otherthan asparagine for asparagine at position 191 (N191) of SEQ ID NO: 1,substituting a protein comprising an amino acid other than threonine orserine for threonine at position 193 (T193) of SEQ ID NO: 1, or both.29. A composition comprising: the recombinant Factor X according toclaim 28; and a physiologically acceptable excipient.
 30. A method oftreating hemophilia, wherein the method comprises administering to asubject in need thereof an effective amount of the composition accordingto claim
 29. 31. The recombinant Factor X with inhibited glycosylationaccording to claim 24, wherein the recombinant Factor X is obtained by aprocess comprising inhibiting glycosylation of asparagine at position181 (N181) and position 191 (N191) of SEQ ID NO: 1 by: substituting aprotein comprising an amino acid other than asparagine for asparagine atposition 181 (N181) of SEQ ID NO: 1, substituting a protein comprisingan amino acid other than threonine or serine for threonine at position183 (T183) of SEQ ID NO: 1, or both; and substituting a proteincomprising an amino acid other than asparagine for asparagine atposition 191 (N191) of SEQ ID NO: 1, substituting a protein comprisingan amino acid other than threonine or serine for threonine at position193 (T193) of SEQ ID NO: 1, or both.
 32. A composition comprising: therecombinant Factor X according to claim 31; and a physiologicallyacceptable excipient.
 33. A method of treating hemophilia, wherein themethod comprises administering to a subject in need thereof an effectiveamount of the composition according to claim
 32. 34. A process forpreparing a recombinant Factor X with inhibited glycosylation, whereinthe process comprises inhibiting glycosylation of threonine at position159 (T159) of SEQ ID NO: 1, inhibiting glycosylation of threonine atposition 443 (T443) of SEQ ID NO: 1, or inhibiting glycosylation ofthreonine at position 159 (T159) and position 443 (T443) of SEQ IDNO:
 1. 35. The process according to claim 34, wherein the processcomprises inhibiting glycosylation of threonine at position 159 (T159)of SEQ ID NO:
 1. 36. The process according to claim 35, wherein theprocess comprises substituting an amino acid other than threonine orserine for threonine at position 159 (T159) of SEQ ID NO:
 1. 37. Theprocess according to claim 35, wherein the process comprisessubstituting a protein comprising an amino acid other than threonine orserine for threonine at position 159 (T159) of SEQ ID NO:
 1. 38. Theprocess according to claim 34, wherein the process comprises inhibitingglycosylation of threonine at position 443 (T443) of SEQ ID NO:
 1. 39.The process according to claim 38, wherein the process comprisessubstituting an amino acid other than threonine or serine for threonineat position 443 (T443) of SEQ ID NO:
 1. 40. The process according toclaim 38, wherein the process comprises substituting a proteincomprising an amino acid other than threonine or serine for threonine atposition 443 (T443) of SEQ ED NO:
 1. 41. The process according to claim34, wherein the process comprises inhibiting glycosylation of threonineat position 159 (T159) and position 443 (T443) of SEQ ID NO:
 1. 42. Theprocess according to claim 41, wherein the process comprisessubstituting an amino acid other than threonine or serine for threonineat position 159 (T159) and position 443 (T443) of SEQ ID NO:
 1. 43. Theprocess according to claim 41, wherein the process comprisessubstituting a protein comprising an amino acid other than threonine orserine for threonine at position 159 (T159) and position 443 (T443) ofSEQ ID NO:
 1. 44. A recombinant Factor X with inhibited glycosylation,wherein the recombinant Factor X is obtained by a process comprisinginhibiting glycosylation of threonine at position 159 (T159) of SEQ IDNO: 1, inhibiting glycosylation of threonine at position 443 (T443) ofSEQ ID NO: 1, or inhibiting glycosylation of threonine at position 159(T159) and position 443 (T443) of SEQ ID NO:
 1. 45. The recombinantFactor X with inhibited glycosylation according to claim 44, wherein therecombinant Factor X is obtained by a process comprising inhibitingglycosylation of threonine at position 159 (T159) of SEQ ID NO: 1 bysubstituting an amino acid other than threonine or serine for threonineat position 159 (T159) of SEQ ID NO: 1, substituting a proteincomprising an amino acid other than threonine or serine for threonine atposition 159 (T159) of SEQ ID NO: 1, or both.
 46. A compositioncomprising: the recombinant Factor X according to claim 45; and aphysiologically acceptable excipient.
 47. A method of treatinghemophilia, wherein the method comprises administering to a subject inneed thereof an effective amount of the composition according to claim46.
 48. The recombinant Factor X with inhibited glycosylation accordingto claim 44, wherein the recombinant Factor X is obtained by a processcomprising inhibiting glycosylation of threonine at position 443 (T443)of SEQ ID NO: 1 by substituting an amino acid other than threonine orserine for threonine at position 443 (T443) of SEQ ID NO: 1,substituting a protein comprising an amino acid other than threonine orserine for threonine at position 443 (T443) of SEQ ID NO: 1, or both.49. A composition comprising: the recombinant Factor X according toclaim 48; and a physiologically acceptable excipient.
 50. A method oftreating hemophilia, wherein the method comprises administering to asubject in need thereof an effective amount of the composition accordingto claim
 49. 51. The recombinant Factor X with inhibited glycosylationaccording to claim 44, wherein the recombinant Factor X is obtained by aprocess comprising inhibiting glycosylation of threonine at position 159(T159) and position 443 (T443) of SEQ ID NO: 1 by: substituting an aminoacid other than threonine or serine for threonine at position 159 (T159)of SEQ ID NO: 1, substituting a protein comprising an amino acid otherthan threonine or serine for threonine at position 159 (T159) of SEQ IDNO: 1, or both; and substituting an amino acid other than threonine orserine for threonine at position 443 (T443) of SEQ ID NO: 1,substituting a protein comprising an amino acid other than threonine orserine for threonine at position 443 (T443) of SEQ ID NO: 1, or both.52. A composition comprising: the recombinant Factor X according toclaim 51; and a physiologically acceptable excipient.
 53. A method oftreating hemophilia, wherein the method comprises administering to asubject in need thereof an effective amount of the composition accordingto claim 52.